[方法] 判斷細胞(eukaryotic cell)培養是否污染
[Please do not crosspost in any format!]
以下摘錄自
Kathy Barker: At the Bench- A Laboratory Navigator
Cold Spring Harbor Laboratory Press
Chapter: "Eukaryotic Cell Culture " (p.231-233)
☆ 巨觀檢查：拿起你的flask or dish對準光線；check for
1.) Cloudiness: "Even in a dense culture, the medium should be clear"
2.) Medium Color Change:
phenol red pH indicator in red medium will turn yellow
in acidic condition; magenta in alkaline condition
bacterial infection often turn medium yellow
fungal contamination may turn it hot pink
3.) Smell:
某些污染會散出惡臭
☆ 微觀檢查：先以10X物鏡鎖定範圍，後以40X物鏡深入檢查（total 400X放大倍率）；
並需觀察不同景深（depth）
[這段需要參考作者畫的污染圖]
(低倍數即可觀察)
fungi污染: long strand, often across the field
yeast污染：visible and appear to be smooth balls, sometimes budding
被破壞的細胞： bacterial infection can kill cells. cell become more
irregularly shaped, larger or smaller; with a dark granularity visible at
low power.
(高倍數才可被觀察)
bacteria：
may be rods or cocci, singly or in chains or in clumps.
They may be associate with cells, or within cells.
May be motile.
The bacteria may be so many that the cell are obscured. There may be only one
contaminant every two fields, which still counts as contamination.
☆ special note for suspension cell:（原文照貼）
Harder (to examine) than adherent cells, especially at high power.
1.) after you place the flask on the microscope stage, leave it for a minute
to let the turbulence slow
2.) focus on the bottom of the flask, and look carefully for slightly adherent
cells that may have attached microorganisms.
3.) focus up through the flask, and whenever you have cells in focus, use
your fine focus knob to examine around the cells for contamination
☆ 診斷污染存在的bioassay
A.) 將medium離心, recover in 50 ul buffer or medium
做methylene blue or Grant stain
B.) streak some of the cell/medium on a blood agar plate and incubate at 37C
for 3 days, colonies? --> contamination!
C.) remove 1 ml of cells/medium to a sterile microfuge tube and incubate for
3 days in the incubator. Observe for cloudiness and make a wet mount.
--
Data speaks louder than words.
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◆ From: 61.229.171.93
※ 編輯: chiuj 來自: 61.229.171.93 (02/14 01:23)
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